Marker for health assessment, and application thereof

ABSTRACT

A marker for health assessment, and an application thereof, relating to the field of medical test. An application of a marker identification reagent in preparation of a reagent for measuring the health of a mammal, comprising: (1) measuring a marker in a test sample, wherein the marker is the level of 8-oxoGsn; and (2) comparing the marker with a reference, wherein the difference between the marker and the reference is used for assessing the health condition of the subject. According to the invention, comprehensive health condition assessment is performed on a subject by means of a simple clinical examination method, and the health condition and health laws of people can be obtained timely and quickly; compared with conventional medical test methods, the invention has remarkable advantages in detection and early warning of sub-health, disease treatment effect monitoring, and psychological emotional state detection in the field of health service management.

FIELD OF THE INVENTION

The present invention relates to a marker for health assessment and uses thereof, more specifically to a use of ratio of free 8-oxoGsn/creatinine in urine as a new health assessment indicator in clinic (i.e., using the ratio of them as an assessment indicator), and belongs to the field of medical examination.

BACKGROUND OF THE INVENTION

Lifestyles, environmental factors and diseases affect people's health all the time. Due to the lack of physical exercise, diseases such as cardiovascular, cerebrovascular, gastrointestinal system, immune system and cervical spine diseases may occur; using computer for a long time may cause vision loss, joint damage, radiation injury, head and shoulder pain, autonomic nerve disorder, depression, arteriosclerotic psychosis, etc.; water pollution, air pollution, automobile exhaust pollution and the like are harmful to the human body, and long-term use of food additives, antibiotics and the like will also adversely affect physical health.

At present, the assessment of individual health status is mostly limited to the scope of medical tests, and multiple medical tests are generally required to obtain the evaluation results of overall health status of the tested individual, which have the disadvantages of time-consuming and high testing costs, and are not suitable for the rapid analysis of large quantities of samples, nor for diagnosis and assessment of overall health. In addition, there is a lack of clinical indicators to indicate whether an individual is in an unhealthy status when the body does not show a “sub-healthy” status with pathological changes, such as fatigue, weak, decreased immunity and resistance, easy colds, shortness of breath during exercise, sweating, backache, leg pain, loss of appetite and so on, as well as mental malaise, depressed mood, sluggish response, insomnia and dreaminess, attention-deficit disorder, memory loss, dysphoria, anxiety, and frightening, etc.

Recent studies have shown that oxidative damage to RNA is a new pathogenic mechanism that plays an important role in aging, diabetes and other diseases, and has received more and more attention. 8-Oxidized guanosine (8-oxo-Gsn) is an oxidation product of guanosine in RNA and can be used as a biomarker to indicate the level of RNA oxidative damage in the body. Compared to other tissues and body fluid samples, 8-oxo-Gsn in urine changes more rapidly after oxidative damage and is more easily detected. Chinese patent ZL 201110309531 discloses a method for detecting 8-oxoGsn (8-oxo-Gsn) in urine of a mammal, in which the kit, detection system and detection method of the invention can quickly and easily determine the concentration of 8-oxoGsn, showing good specificity, high sensitivity, accurate and reliable results. The 8-oxoGsn detection technology of the invention is currently an advanced biological information detection technology in the world, and has been successfully applied to the evaluation of human aging degree and early warning of aging-related diseases.

Given the high sensitivity of 8-oxoGsn and the relevance of increased level thereof to various diseases, it is necessary to study the relationship between 8-oxoGsn level in urine and diseases. At present, there is no report on the relationship between the interval of the ratio of 8-oxoGsn concentration to creatinine concentration and the health status, and existing studies have not shown that 8-oxoGsn can be used as a health marker.

SUMMARY OF THE INVENTION

The problem to be solved by the present invention is to provide a novel health condition marker for a mammal (including but not limited to a human), and a reagent for detecting the marker.

The present invention has been completed based on the following findings of the applicant, that is, there is a detectable and evaluable correspondence between the ratio of 8-oxoGsn concentration to creatinine concentration (8-oxoGsn/creatinine) in a body fluid of a mammal (including but not limited to a human) and the health status of the mammal, and this ratio can be used as a new marker of mammalian health.

To achieve the above purpose, the present invention adopts the following technical solutions.

A first aspect of the present invention provides a use of a reagent for identifying a marker in manufacture of a reagent for detecting a health status of a mammal, comprising:

(1) detecting a marker in a sample of a subject, wherein the marker is a ratio of 8-oxoGsn concentration to creatinine concentration;

(2) comparing the marker with a reference, wherein a difference of the marker compared with the reference is used to evaluate a health status of the subject.

The sample is a body fluid.

The body fluid includes peripheral blood, serum, plasma, synovial fluid, aqueous humor, breast milk, semen, prostate fluid, sweat, tear, pleural effusion, ascites fluid, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, vomitus, vaginal secretion, mucosal secretion, pancreatic juice, bronchopulmonary suction fluid, blastocyst cavity fluid, umbilical cord blood, urine, cerebrospinal fluid, saliva, lymph fluid and excreta.

The body fluid is preferably urine, peripheral blood, serum, or plasma. Most preferred is urine of a mammal.

For a plasma sample, proteins are first precipitated with acetonitrile, the supernatant is blow dried with nitrogen, redissolved with a mobile phase, and the amount of 8-oxoGsn is detected by HPLC-MS/MS. The concentration of 8-oxoGsn is directly derived from an 8-oxoGsn standard curve.

For a blood sample, HPLC eluent is usually used in gradient elution to obtain better separation results. But this necessarily increases the complexity of detection. At the same time, the treatment of sample must be more complicated, resulting in longer testing time and increased cost. Therefore, from the perspective of convenience and economy, a urine sample is most preferred.

The level of 8-oxoGsn is the ratio of 8-oxoGsn concentration to creatinine concentration in a sample. The creatinine concentration of urine sample is measured simultaneously to correct the effect of drinking water on the 8-oxoGsn concentration in urine.

The reference is any one or both of the following:

(1) an interval value of 8-oxoGsn level of a healthy mammal;

(2) an interval value of ratio of 8-oxoGsn concentration to creatinine concentration of a healthy mammal.

An interval value is 95% percentile. The 0-95 percentile of healthy subjects is used as a normal reference interval.

The mammal according to the present invention is preferably a human, but may also be other mammal, such as cat, dog, horse, and the like.

The method for determining 8-oxoGsn in a mammal belongs to the prior art. According to a preferred embodiment, an 8-oxoGsn standard is first used to prepare a standard curve, so that the concentration of 8-oxoGsn in a sample can be determined more quickly and accurately. The concentration range of the standard curve can be adjusted according to the different samples to be measured.

According to a more preferred embodiment, an internal standard is added to the sample to be measured in order to correct the error of measuring instrument. The internal standard may be 8-oxoGsn in which any one or more elements are replaced by isotopes, and preferably at least one of N and C is replaced by their corresponding isotopes. For example, ¹⁴N can be replaced by ¹⁵N, and ¹²C can be replaced by ¹³C. It is possible to replace only one element, or replace two or more elements. In one test, the concentration of internal standard in each sample is the same.

A second aspect of the present invention provides a use of a reagent for detecting 8-oxoGsn in manufacture of a product (including but not limited to a kit) for evaluating a health status of a mammal.

The reagent for detecting 8-oxoGsn is a reagent for determining the concentration of 8-oxoGsn.

The mammal is a human.

A third aspect of the present invention provides a use of a reagent for detecting 8-oxoGsn concentration and creatinine concentration in manufacture of a product (including but not limited to a kit) for evaluating a health status of a mammal.

By using the kit, the 8-oxoGsn concentration and creatinine concentration in a sample of a subject are measured, the ratio of the 8-oxoGsn concentration to the creatinine concentration is calculated, and the ratio is compared with a reference to evaluate the health status of the subject who is the source of the sample to be tested.

The reference is an interval (95% percentile) of 8-oxoGsn level in a healthy mammal.

The reference is an interval (95% percentile) of the ratio of 8-oxoGsn concentration to creatinine concentration in a healthy mammal.

A fourth aspect of the present invention provides a kit for detecting a health status of a mammal, comprising a reagent for detecting 8-oxoGsn concentration and a reagent for detecting creatinine concentration.

The mammal is a human.

The kit comprises 8-oxoGsn standard, 10 mM ammonium acetate, methanol, 1 to 2000 pg/μL isotope internal standard [¹³C,¹⁵N₂]8-oxo-dGsn, and 1 to 2000 pg/μL isotope internal standard [¹⁵N₅]8-oxoGsn; preferably, the isotope internal standards have a concentration of 100 to 800 pg/μL.

The 10 mM ammonium acetate and methanol are 70% methanol+30% 10 mM ammonium acetate solution, and the solution after mixing has a pH of 3.5 to 4.0. That is, the working solution in this kit is prepared from methanol and 10 mM ammonium acetate solution in a volume ratio of 7:3.

We established a reference interval of 8-oxoGsn for urine samples from normal population in Beijing, China by using the isotope dilution high performance liquid chromatography mass spectrometry technology (the method was the same as Chinese patent ZL201110309531.5, “Application of kit, detection system and 8-oxoGsn for assessment of aging degree of mammals”), as shown in FIG. 1 and Table 1. By studying a large size of samples, it was found that normal humans between the ages of 20 and 74, regardless of gender, had a positive correlation between the 8-oxoGsn content in their urine and their age, which could be used as an age-related indicator.

By using the same method, we determined 8-oxoGsn in urine samples of patients of seven diseases (AIDS, hepatitis B, hepatitis C, syphilis, liver cancer, coronary heart disease, acute coronary syndrome), compared the results of the seven diseases with the above reference interval of 8-oxoGsn for normal humans, and found that the 8-oxoGsn contents in the urine samples of the patients of various diseases increased significantly as compared with the normal humans. See Table 2 and FIG. 2 for details. At least 60% of the patients of each disease had higher values than the interval for normal humans. In particular, for the bacterial and viral infectious diseases, the proportion of number of the patients having a value higher than the interval for normal humans was as high as about 90%, while chronic diseases such as syphilis, AIDS, hepatitis B, etc., come next, which proved that the increase of 8-oxoGsn was correlated with diseases.

Further, we recruited 8 healthy volunteers aged 20-30, each of them had to collect urine 25 times within 2 months, and each time the urine was collected with a urine cup, immediately transferred to a 5 ml urine tube and stored in a refrigerator at −80° C., and the physical conditions of the day, such as whether there was strenuous exercise, physical discomfort, insomnia, high work intensity and other factors, were recorded in the corresponding record manual (see Table 3 for details), which were used to screen the specific influencing factors causing the increase of 8-oxoGsn level in normal humans. When the final test results of each person's urine samples were compared with the corresponding upper limit of the 95th percentile of the reference interval for normal humans, it was found that most of the time points where the values were above the upper limit of the interval for normal humans corresponded to the special statuses of the day, such as illness, excessive fatigue, staying up late, strenuous exercise, etc. The above results fully reflect the high sensitivity of the 8-oxoGsn level in urine, that is, as long as a normal healthy person feels an uncomfortable symptom, especially a state of disease, whose level would increase correspondingly, so that this may reflect a health status of a human to a certain extent. In addition, it was found in this experiment that the level of RNA oxidation in a human who exercised regularly was significantly lower than that in those who did not exercise, for example, as shown in FIG. 3, the healthy humans Nos. 201 and 202 basically maintained their 8-oxoGsn levels in urine below the upper limit of the 95th percentile of the reference interval for normal humans.

In addition, we also tested the level of nucleic acid oxidation in the plasma and urine of patients with chronic kidney disease (CKD). From Table 4, it could be found that the level of RNA oxidation was positively correlated with the severity of the disease, so that the increased 8-oxoGsn ratio in plasma and urine might be a new assessment indicator for advanced kidney disease.

Through a large number of experimental studies, the present invention obtained the value interval of 8-oxoGsn in subjects of different ages at health status, and corresponding data at different health statuses such as psychological stress status, sub-health status, chronic disease status, acute disease status, cancerous disease status, viral disease status and the like. Through scientific and in-depth analysis and research on these data, the present invention has found for the first time the correlation between 8-oxoGsn and the overall health of human, in which the value of 8-oxoGsn and the health status show a significant positive correlation, so that a valuable health status assessment of a subject can be carried out by only measuring 8-oxoGsn of the body. The above research results show that the 8-oxoGsn in urine is not only a useful indicator for evaluating oxidative stress and aging of the body, but also a new clinical health evaluation indicator.

The present invention has wide application prospects, and can be used for detection and early warning of diseases, psychological evaluation of students, troops and special working groups, supporting detection of the disease status of tumor patients and so on, and is particularly suitable for epidemiological censuses, for health screening of humans at a certain area or a certain age group, for assessment of effects of external factors on the health status of the population, and for guiding people to change their lifestyles or receive medical services to improve their health. The present invention adopts a simple clinical physical examination method to conduct a comprehensive health status assessment on a subject, can timely and quickly grasp people's health status and health rules, can be used in the field of health service management for sub-health detection and early warning, disease treatment and effect observation, and detection of psychological and emotional state, and have significant advantages over conventional medical detection methods.

The following describes the present invention in detail with reference to the examples and the drawings, but is not a limitation on the present invention. Any equivalent replacement in the art made in accordance with the disclosure of the present invention belongs to the protection scope of the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the creatinine-corrected 8-oxoGsn/cre values in the urine samples of normal males and females aged 20-74 years.

FIG. 2A shows a comparison of the urine 8-oxoGsn/cre level of male patients with HIV with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2B shows a comparison of the urine 8-oxoGsn/cre level of male patients with hepatitis B with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2C shows a comparison of the urine 8-oxoGsn/cre level of female patients with hepatitis B with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2D shows a comparison of the urine 8-oxoGsn/cre level of male patients with hepatitis C with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2E shows a comparison of the urine 8-oxoGsn/cre level of female patients with hepatitis C with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2F shows a comparison of the urine 8-oxoGsn/cre level of male patients with syphilis with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2G shows a comparison of the urine 8-oxoGsn/cre level of female patients with syphilis with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2H shows a comparison of urine 8-oxoGsn/cre levels of male patients with ACS with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2I shows a comparison of the urine 8-oxoGsn/cre level of female patients with ACS with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2J shows a comparison of the urine 8-oxoGsn/cre level of male patients with coronary heart disease with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2K shows a comparison of the urine 8-oxoGsn/cre level of female patients with coronary heart disease with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2L shows a comparison of the urine 8-oxoGsn/cre level of male patients with liver cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2M shows a comparison of the urine 8-oxoGsn/cre level of female patients with liver cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2N shows a comparison of the urine 8-oxoGsn/cre level of male patients with kidney cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2O shows a comparison of the urine 8-oxoGsn/cre level of female patients with kidney cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2P shows a comparison of the urine 8-oxoGsn/cre level of male patients with bladder cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2Q shows a comparison of the urine 8-oxoGsn/cre levels of female patients with bladder cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 2R shows a comparison of the urine 8-oxoGsn/cre level of male patients with prostate cancer with the upper limit of the 95th percentile of the reference interval for normal humans.

FIG. 3A to 3H show comparisons the 8-oxoGsn/cre levels in 25 urine samples collected from 8 normal humans within 2 months with the upper limit of the 95th percentile of the reference interval for normal humans.

DETAILED DESCRIPTION OF THE INVENTION Embodiment 1: Determination of 8-oxoGsn Content in Urine of Normal Humans Aged 20-74 Years

I. Reagents and Methods:

(1) Samples: 5179 urine samples from normal humans, including 2591 males and 2588 females.

(2) Reagents and instruments:

Reagents: 8-oxidized guanosine (purity>98%), purchased from ALEXIS Company, the United States; 8-oxidized deoxyguanosine (purity>98%), purchased from Sigma-Aldrich, the United States; [¹³C, ¹⁵N₂]8-oxidized guanosine (purity>98%), purchased from Toronto Research Chemical Company, Canada; [¹⁵N₅]8-oxidized deoxyguanosine (purity>98%), purchased from Cambridge Isotope Laboratory, the United States; creatinine detection kit, purchased from Beijing Jiuqiang Biotechnology Company, China; methanol (HPLC grade), purchased from Merck, Germany; isopropanol (HPLC grade), purchased from Merck, Germany; formic acid (HPLC grade), purchased from Merck, Germany; ammonium acetate (HPLC grade), purchased from Fisher Technology, USA;

Chromatography column: Agilent SB-Aq, 3.0×100 mm, 1.8 μm

Instrument: the instrument used for HPLC-MS/MS analysis was an Agilent LC (1290) tandem mass spectrometer (6490) instrument.

(3) Determination method:

3 ml of middle urine in random urine was collected, evenly divided into 3 parts and placed into 1.5 ml EP tubes, and stored in a refrigerator at −80° C. until testing. The treatment of urine samples was as follows:

1. A urine sample was thawed and centrifuged at 7,500 g for 5 min at 4° C.

2. 200 μL of the supernatant was pipetted into a 1.5 ml autoclaved centrifuge tube. 200 μL of working solution A [70% methanol+30% 10 mM ammonium acetate (PH=3.7)] was added; and the working solution was used to precipitate proteins present in the urine.

3. 480 pg/μL isotope internal standard [¹³C, ¹⁵N₂]8-oxo-dGsn and 480 pg/μL isotope internal standard [¹⁵N₅]8-oxoGsn, each 10 μL, were added, mixed by votex for 2 min; the internal standards were used for the correction of measuring instrument errors.

4. After being placed in a 37° C. biochemical incubator for 10 minutes, centrifugation was carried out at 12000 g and 4° C. for 15 minutes; this step allowed the 8-oxoG combined with the precipitate to be released.

5. 100 ul was taken and added into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

6. The standard 8-oxoG was detected by HPLC-MS/MS, and a standard curve of 8-oxoG concentration versus intensity was prepared. At the same time, the intensity of 8-oxoG in the sample was detected by HPLC-MS/MS, and the concentration of 8-oxoG in the sample was calculated against the standard curve.

7. The creatinine concentration in the sample was measured with a Hitachi biochemical detector; which was used to correct the effect of different dilution levels of urine on the results.

II. Results

See FIG. 1 and Table 1 (creatinine-corrected mean values and percentiles of 8-oxoGsn in the reference population). A large sample study found that, for normal humans aged 20 to 74, regardless of gender, the 8-oxoGsn content had a positive correlation with the age, and could be used as an age-related indicator. Compared with normal humans, the level of 8-oxoGsn in patients with diseases was higher than that in normal humans, and the higher values, the more meaningful. Therefore, the 0-95 percentile of each age group was used as the reference interval for normal humans (see Table 1 for 0-95% reference interval for each age group).

Embodiment 2: Determination of 8-oxoGsn Content in Urine of Patients of Seven Diseases

I. Reagents and Methods:

(1) Samples: 111 urine samples from AIDS patients, 115 urine samples from hepatitis B patients, 104 urine samples from hepatitis C patients, 79 urine samples from syphilis patients, 90 urine samples from liver cancer patients, 119 urine samples from patients with coronary heart disease, and 98 urine samples from ACS patients were collected.

(2) Reagents and instruments:

Reagents: 8-oxidized guanosine (purity>98%), purchased from ALEXIS Company, the United States; 8-oxidized deoxyguanosine (purity>98%), purchased from Sigma-Aldrich, the United States; [¹³C, ¹⁵N₂]8-oxidized guanosine (purity>98%), purchased from Toronto Research Chemical Company, Canada; [¹⁵N₅]8-oxidized deoxyguanosine (purity>98%), purchased from Cambridge Isotope Laboratory, the United States; creatinine detection kit, purchased from Beijing Jiuqiang Biotechnology Company, China; methanol (HPLC grade), purchased from Merck, Germany; isopropanol (HPLC grade), purchased from Merck, Germany; formic acid (HPLC grade), purchased from Merck, Germany; ammonium acetate (HPLC grade), purchased from Fisher Technology, USA;

Chromatography column: Agilent SB-Aq, 3.0×100 mm, 1.8 μm

Instrument: the instrument used for HPLC-MS/MS analysis was an Agilent LC (1290) tandem mass spectrometer (6490) instrument.

(3) Determination method:

3 ml of middle urine in random urine was collected, evenly divided into 3 parts and placed into 1.5 ml EP tubes, and stored in a refrigerator at −80° C. until testing. The treatment of urine samples was as follows:

1. A urine sample was thawed and centrifuged at 7,500 g for 5 min at 4° C.

2. 200 μL of the supernatant was pipetted into a 1.5 ml autoclaved centrifuge tube. 200 μL of working solution A [70% methanol+30% 10 mM ammonium acetate (PH=3.7)] was added; and the working solution was used to precipitate proteins present in the urine.

3. 480 pg/μL isotope internal standard [¹³C, ¹⁵N₂]8-oxo-dGsn and 480 pg/μL isotope internal standard [¹⁵N₅]8-oxoGsn, each 10 μL, were added, mixed by votex for 2 min; the internal standards were used for the correction of measuring instrument errors.

4. After being placed in a 37° C. biochemical incubator for 10 minutes, centrifugation was carried out at 12000 g and 4° C. for 15 minutes; this step allowed the 8-oxoG combined with the precipitate to be released.

5. 100 ul was taken and added into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

6. The standard 8-oxoG was detected by HPLC-MS/MS, and a standard curve of 8-oxoG concentration versus intensity was prepared. At the same time, the intensity of 8-oxoG in the sample was detected by HPLC-MS/MS, and the concentration of 8-oxoG in the sample was calculated against the standard curve.

7. The creatinine concentration in the sample was measured with a Hitachi biochemical detector; which was used to correct the effect of different dilution levels of urine on the results.

II. Results

The results were shown in FIG. 2 and Table 2 (the numbers and percentages of patients with various diseases that were of above the 95th percentile of the reference interval for normal humans), and for the 8-oxoGsn in urine samples of patients with seven diseases (AIDS, hepatitis B, hepatitis C, syphilis, liver cancer, coronary heart disease, acute coronary syndrome), a comparative study was performed between the results of the seven diseases and the reference interval of 8-oxoGsn for normal humans, and it was found that the levels of 8-oxoGsn in urine samples of patients with various diseases were significantly increased compared to that of the normal humans (see Table 2 and FIG. 2 for details), and at least 60% of the patients of each disease had higher values than the interval for normal humans. In particular, for the bacterial and viral infectious diseases, the proportion of number of patients having a value higher than the interval for normal humans was as high as about 90%, while chronic diseases such as syphilis, AIDS, hepatitis B, etc., come next, which proved that the increase of 8-oxoGsn was correlated with the diseases.

Embodiment 3: Determination of 8-oxoGsn Content in Urine of Healthy Volunteers

I. Reagents and Methods:

(I) Samples: collected from a total of 8 healthy volunteers, and urine was collected for 25 times from each of the volunteers.

(2) Reagents and instruments:

Reagents: 8-oxidized guanosine (purity>98%), purchased from ALEXIS Company, the United States; 8-oxidized deoxyguanosine (purity>98%), purchased from Sigma-Aldrich, the United States; [¹³C, ¹⁵N₂]8-oxidized guanosine (purity>98%), purchased from Toronto Research Chemical Company, Canada; [¹⁵N₅]8-oxidized deoxyguanosine (purity>98%), purchased from Cambridge Isotope Laboratory, the United States; creatinine detection kit, purchased from Beijing Jiuqiang Biotechnology Company, China; methanol (HPLC grade), purchased from Merck, Germany; isopropanol (HPLC grade), purchased from Merck, Germany; formic acid (HPLC grade), purchased from Merck, Germany; ammonium acetate (HPLC grade), purchased from Fisher Technology, USA;

Chromatography column: Agilent SB-Aq, 3.0×100 mm, 1.8 μm

Instrument: the instrument used for HPLC-MS/MS analysis was an Agilent LC (1290) tandem mass spectrometer (6490) instrument.

(3) Determination method:

3 ml of middle urine in random urine was collected, evenly divided into 3 parts and placed into 1.5 ml EP tubes, and stored in a refrigerator at −80° C. until testing. The treatment of urine samples was as follows:

1. A urine sample was thawed and centrifuged at 7,500 g for 5 min at 4° C.

2. 200 μL of the supernatant was pipetted into a 1.5 ml autoclaved centrifuge tube. 200 μL of working solution A [70% methanol+30% 10 mM ammonium acetate (PH=3.7)] was added; and the working solution was used to precipitate proteins present in the urine.

3. 480 pg/μL isotope internal standard [¹³C, ¹⁵N₂]8-oxo-dGsn and 480 pg/μL isotope internal standard [¹⁵N₅]8-oxoGsn, each 10 μL, were added, mixed by votex for 2 min; the internal standards were used for the correction of measuring instrument errors.

4. After being placed in a 37° C. biochemical incubator for 10 minutes, centrifugation was carried out at 12000 g and 4° C. for 15 minutes; this step allowed the 8-oxoG combined with the precipitate to be released.

5. 100 ul was taken and added into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

6. The standard 8-oxoG was detected by HPLC-MS/MS, and a standard curve of 8-oxoG concentration versus intensity was prepared. At the same time, the intensity of 8-oxoG in the sample was detected by HPLC-MS/MS, and the concentration of 8-oxoG in the sample was calculated against the standard curve.

7. The creatinine concentration in the sample was measured with a Hitachi biochemical detector; which was used to correct the effect of different dilution levels of urine on the results.

II. Results

The results were shown in FIG. 3 and Table 3 (the physical discomfort records of the 8 healthy volunteers on the days for collecting 25 urine samples were assessed), and when the final test results of each volunteer's urine were compared with the corresponding upper limit of the 95th percentile of the reference interval for normal humans, it was found that most of the time points where the values were above the upper limit of the interval for normal humans corresponded to the special statuses of the day, such as illness, excessive fatigue, staying up late, strenuous exercise and the like. The above results fully reflected the high sensitivity of the level of 8-oxoGsn in urine, that was, as long as a normal healthy person felt an uncomfortable symptom, especially a state of disease, whose level would increase correspondingly, so that this could reflect a health status of a human to a certain extent. In addition, it was found in this experiment that the level of RNA oxidation in a human who exercised regularly was significantly lower than those who did not exercise, for example, as shown in FIG. 3, the healthy humans Nos. 201 and 202 basically maintained their 8-oxoGsn levels in urine below the upper limit of the 95th percentile of the reference interval for normal humans.

Embodiment 4

I. Reagents and Methods:

(I) Test population and sample size: plasma and urine samples were collected from a total of 146 patients with chronic kidney diseases, which included 30 patients with phase I chronic kidney disease, 30 patients with phase II chronic kidney disease, 31 patients with phase III chronic kidney disease, 30 patients with phase IV chronic kidney disease, and 25 patients with phase V chronic kidney disease.

(2) Reagents and instruments, comprising:

Reagents: 8-oxidized guanosine (purity>98%), purchased from ALEXIS Company, the United States; 8-oxidized deoxyguanosine (purity>98%), purchased from Sigma-Aldrich, the United States; [¹³C, ¹⁵N₂]8-oxidized guanosine (purity>98%), purchased from Toronto Research Chemical Company, Canada; [¹⁵N₅]8-oxidized deoxyguanosine (purity>98%), purchased from Cambridge Isotope Laboratory, the United States; creatinine detection kit, purchased from Beijing Jiuqiang Biotechnology Company, China; methanol (HPLC grade), purchased from Merck, Germany; isopropanol (HPLC grade), purchased from Merck, Germany; formic acid (HPLC grade), purchased from Merck, Germany; ammonium acetate (HPLC grade), purchased from Fisher Technology, USA;

Chromatography column: Agilent SB-Aq, 3.0×100 mm, 1.8 μm

Instrument: the instrument used for HPLC-MS/MS analysis was an Agilent LC (1290) tandem mass spectrometer (6490) instrument.

(3) Determination method: urine and/or plasma samples could be selected for detection

Detection of urine sample: 3 ml of middle urine in random urine was collected, evenly divided into 3 parts and placed into 1.5 ml EP tubes, and stored in a refrigerator at −80° C. until testing. The treatment of urine samples was as follows: 1. A urine sample was thawed and centrifuged at 7,500 g for 5 min at 4° C.

2. 200 μL of the supernatant was pipetted into a 1.5 ml autoclaved centrifuge tube. 200 μL of working solution A [70% methanol+30% 10 mM ammonium acetate (PH=3.7)] was added; and the working solution was used to precipitate proteins present in the urine.

3. 480 pg/μL isotope internal standard [¹³C, ¹⁵N₂]8-oxo-dGsn and 480 pg/μL isotope internal standard [¹⁵N₅]8-oxoGsn, each 10 μL, were added, mixed by votex for 2 min; the internal standards were used for the correction of measuring instrument errors.

4. After being placed in a 37° C. biochemical incubator for 10 minutes, centrifugation was carried out at 12000 g and 4° C. for 15 minutes; this step allowed the 8-oxoG combined with the precipitate to be released.

5. 100 ul was taken and added into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

6. The standard 8-oxoG was detected by HPLC-MS/MS, and a standard curve of 8-oxoG concentration versus intensity was prepared. At the same time, the intensity of 8-oxoG in the sample was detected by HPLC-MS/MS, and the concentration of 8-oxoG in the sample was calculated against the standard curve.

7. The creatinine concentration in the sample was measured with a Hitachi biochemical detector; which was used to correct the effect of different dilution levels of urine on the results.

Determination of plasma samples: 3 ml of plasma was collected from a patient, evenly aliquoted and loaded into 1.5 ml EP tubes, and stored at −80° C. in a refrigerator until testing. The treatment of plasma samples was as follows:

1. The collected plasma was taken from the −80° C. refrigerator and thawed on ice, mixed well and centrifuged at 14000 g and 4° C. for 10 minutes.

2. 300 μL was taken and added with two internal standards, 300 pg/μL and 10 μL for each. The internal standards were used to correct errors of the measuring instrument.

3. After being mixed well with three times volume acetonitrile of 900 μL, vortex was performed for approximately 1 minute, so that the proteins present in the plasma were precipitated with acetonitrile.

4. After centrifugation at 14,000 g for 20 minutes at 4° C., a quantitative volume of 1 ml supernatant was taken and blow dried with nitrogen.

5. After being redissolved with 100 μL of mobile phase, centrifugation was carried out at 12000 g for 20 minutes at 4° C.

6. 85 μL was taken and placed into a disposable intubation tube, loaded into a sample bottle, and subjected to mass spectrometry sequence detection.

II. Results

The results were shown in Table 4 (plasma, urine creatinine and nucleic acid oxidation levels of patients with chronic kidney diseases). From Table 4, it could be found that the RNA oxidation level was positively correlated with the severity of the disease, so that the increased 8-oxoGsn ratio in plasma and urine could be a new indicator for advanced kidney diseases.

Embodiment 5: Reagent for Identifying Marker 8-oxoGsn and Use Thereof

I. Kit Composition

1. 8-oxoGsn standard, used to prepare a standard curve of 8-oxoGsn concentration versus intensity.

2. 10 mM ammonium acetate and pure methanol: used to prepare a working solution [70% methanol+30% 10 mM ammonium acetate (PH=3.7)]; which could also be a directly prepared 70% methanol+30% 10 mM ammonium acetate solution (pH was 3.5-4.0), that was, the working solution in this kit was prepared from methanol and 10 mM ammonium acetate solution in a volume ratio of 7:3.

3. 500 pg/μL isotope internal standard [¹³C, ¹⁵N₂]8-oxo-dGsn and 500 pg/μL isotope internal standard [¹⁵N₅]8-oxoGsn.

II. Use for Determining Health Status

1. Detecting a marker in a sample of a subject, wherein the marker was level of 8-oxoGsn;

2. The marker was compared with a reference (the 95% confidence interval value for normal humans obtained in Example 1), and the difference of the marker compared with the reference was used to evaluate the health status of the subject.

3. The following explained how to compare the marker with the reference (the 95% reference interval value for normal humans obtained in Example 1) for evaluating the health status of the subject: among the 25 tests for the urine samples collected from the healthy volunteers, the marker concentrations obtained from the 25 urine tests were compared with the upper limit of the 95% reference interval for normal humans in the age group. Above 95% reference interval indicated that the subject was in a state of high oxidative stress. The concentrations of most or all of the 25 urine markers were higher than the 95% reference interval, indicating that the subject was in an unhealthy state with persistent high oxidative stress, and intervention was needed. Based on the physical discomfort records of the subjects (see Table 3), the effects of exogenous and endogenous pathogenic factors on 8-oxoGsn levels in human urine were identified, and the main factors that caused the increase of 8-oxoGsn level were screened, and real-time assessment of human health was performed to achieve the purpose of early warning of human sub-health and disease.

Although the present invention has been described in detail according to specific embodiments, it should be understood that those skilled in the art can readily conceive of changes, variations, or equivalents of these embodiments after gaining an understanding of the foregoing. Accordingly, the scope of the present invention should be evaluated as that of the appended claims and any equivalents thereof.

TABLE 1 Means and percentiles of 8-oxoGsn in reference population after creatinine correction Male Female Mean ± Mean ± Median standard Median standard Age group No. Creatinine 5th (IQR) 95th deviation No. Creatinine 5th (IQR) 95th deviation 20-24 121 19442(8878) 1.50 1.69(0.14) 1.87  1.7 ± 0.11 121  15219(17118) 1.48 1.68(0.15) 1.82 1.67 ± 0.1  25-29 125 18501(9586) 1.14 1.72(0.45) 2.38  1.7 ± 0.32 124  12996(10642) 1.18 1.60(0.44) 2.00  1.6 ± 0.25 30-34 130 15756(7010) 1.19 1.93(0.37) 2.43 1.89 ± 0.34 130 12518(9591) 1.31 1.86(0.40) 2.29 1.84 ± 0.28 35-39 120 14828(6971) 1.96 2.23(0.19) 2.45 2.23 ± 0.14 131 12005(7572) 1.8 2.04(0.21) 2.32 2.03 ± 0.14 40-44 125 16003(8118) 2.09 2.32(0.15) 2.55 2.33 ± 0.13 122 10777(8401) 1.44 2.12(0.42) 2.55 2.06 ± 0.33 45-49 132 15942(7864) 2.16 2.45(0.2)  2.61 2.43 ± 0.14 133 11229(7000) 2.07 2.31(0.18) 2.56 2.31 ± 0.13 50-54 133 15455(8087) 1.91 2.43(0.27) 2.74 2.38 ± 0.25 127 10058(8086) 2.09 2.49(0.24) 2.61 2.44 ± 0.17 55-59 128  14877(10014) 2.2 2.58(0.17) 2.83 2.55 ± 0.16 137  9542(6640) 2.21 2.51(0.17) 2.70  2.5 ± 0.13 60-64 129 12808(8747) 2.45 2.71(0.15) 2.89 2.68 ± 0.12 120 10355(7945) 2.52 2.71(0.12) 2.90 2.71 ± 0.09 65-69 132 12872(6817) 2.12 2.81(0.35) 3.26 2.75 ± 0.32 125  9643(6428) 2.6 2.77(0.19) 2.94 2.78 ± 0.11 70-74 121 12842(6522) 2.77 3.02(0.18) 3.28 3.02 ± 0.16 128  8934(7056) 2.5 2.84(0.27) 3.11 2.83 ± 0.17 Data were grouped by gender, and the concentration of creatinine was expressed as μmol/L.

TABLE 2 Number and percentage of various diseases above the 95th percentile of the reference interval for normal humans 8-oxoGsn Total Disease Male Female Number number AIDS 90(90%) 90(90%) 100 Hepatitis B 43(85%) 43(85%) 86(86%) 100 Hepatitis C 37(74%) 45(90)    82(82%) 100 Syphilis 37(95%) 38(91%) 75 (93%)  81 Acute coronary syndrome 56(71%) 26(79%) 82(73%) 112 Coronary heart disease 38(75%) 26(52%) 64(64%) 100 Liver cancer 49 (69%)  17 (81%)  66(72%) 92 Kidney cancer 63(76%) 32(70%) 95(74%) 129 Bladder cancer 42(75%)  5 (45%)  47(70%) 67 Prostate 63(72%) 63(72%) 88

TABLE 3 Physical discomfort records on the days of 25 urine health assessments for 8 healthy volunteers Time of collecting Number of normal humans urine sample 201 202 203 205 206 209 211 214 1 Work hard Diarrhea and insomnia 2 Mood Slightly diarrhea, swings strenuous exercise 3 Cold Insomnia Stay-up late 4 Insomnia Insomnia 5 Diarrhea Stay-up late 6 Strenuous exercise 7 Cold and taking Diarrhea Stay-up late cold medicine 8 Cold and taking cold medicine 9 Cold and taking cold medicine 10 Diarrhea Cold and taking cold medicine 11 12 13 14 Insomnia Cold 15 Stay-up late 16 Stay-up late 17 Cold Stay-up late 18 Cold Stay-up late Cold and strenuous exercise 19 Stay-up late Cold 20 Stay-up late 21 Anxiety Stay-up late 22 Anxiety Stay-up late Anxiety and fatigue 23 Strenuous exercise Anxiety Stay-up late Anxiety and fatigue 24 Strenuous exercise Anxiety Stay-up late 25 Strenuous exercise Anxiety Stomach Stay-up late pain

TABLE 4 Plasma, urine creatinine, and nucleic acid oxidation levels in patients with chronic kidney disease CKD 1 CKD 2 CKD 3 CKD 4 CKD 5 Plasma creatinine 61.60 ± 14.21 90.33 ± 26.26 142.87 ± 63.91  202.03 ± 53.21  590.40 ± 277.87 (umol/L) Urine creatinine   12494 ± 6926.21 10270.07 ± 6238.45  7359.17 ± 3882.42 6447.93 ± 4354.35 4268.64 ± 5782.24 (umol/L) Glomerular 132.53 ± 33.21  76.98 ± 9.1  45.81 ± 8.5  24.73 ± 4.56  7.84 ± 2.9  filtration rate Urine 1.87 ± 0.87 1.92 ± 1.01 1.84 ± 0.81 1.47 ± 0.89 1.60 ± 1.59 8-oxo-dGsn/Cr (pmol/mol) Urine 8-oxo-Gsn/Cr (umol/mol) 3.07 ± 1.07 3.42 ± 1.34 3.72 ± 1.47 3.90 ± 1.93 3.75 ± 2.26 Plasma 0.17 ± 0.12 0.24 ± 0.18 0.37 ± 0.20 0.49 ± 0.22 1.10 ± 0.57 8-oxo-Gsn/Cr (pmol/mol) Plasma/urine 0.02 ± 0.02 0.03 ± 0.02 0.06 ± 0.04 0.10 ± 0.05 0.34 ± 0.03 8-oxo-Gsn Data were expressed as mean ± SD; significant differences were considered when P < 0.05 

1. A use of a reagent for identifying a marker in manufacture of a reagent for detecting a health status of a mammal, comprising: (1) detecting a marker in a sample of a subject, wherein the marker is a ratio of 8-oxoGsn concentration to creatinine concentration; (2) comparing the marker with a reference, wherein a difference of the marker compared with the reference is used to evaluate the health status of the subject.
 2. The use according to claim 1, wherein the sample is a body fluid.
 3. The use according to claim 2, wherein the body fluid is selected from the group consisting of peripheral blood, serum, plasma, synovial fluid, aqueous humor, breast milk, semen, prostate fluid, sweat, tear, pleural effusion, ascites fluid, pericardial fluid, chyle, bile, interstitial fluid, menstrual blood, vomitus, vaginal secretion, mucosal secretion, pancreatic juice, bronchopulmonary suction fluid, blastocyst cavity fluid, umbilical cord blood, urine, cerebrospinal fluid, saliva, lymph fluid and excreta.
 4. The use according to claim 3, wherein the body fluid is urine, peripheral blood, serum, or plasma.
 5. The use according to claim 1, wherein the reference is any one or both of the following: (1) an interval value of 8-oxoGsn level of a healthy mammal; (2) an interval value of the ratio of 8-oxoGsn concentration to creatinine concentration of a healthy mammal.
 6. The use according to claim 5, wherein an interval value is 95% percentile.
 7. The use according to claim 1, wherein the mammal is a human.
 8. A use of a reagent for detecting 8-oxoGsn in manufacturing of a product for evaluating a health status of a mammal.
 9. The use according to claim 8, wherein the reagent for detecting 8-oxoGsn is a reagent for determining 8-oxoGsn concentration.
 10. A use of a reagent for detecting 8-oxoGsn concentration and creatinine concentration in manufacturing a product for evaluating a health status of a mammal.
 11. The use according to claim 7, wherein the mammal is a human, and the product is a kit.
 12. A kit for detecting a health status of a mammal, comprising a reagent for detecting 8-oxoGsn concentration and a reagent for detecting creatinine concentration.
 13. The kit according to claim 12, wherein the mammal is a human.
 14. The kit according to claim 12, wherein the kit comprises 8-oxoGsn standard, 10 mM ammonium acetate, methanol, 1 to 2000 pg/μL isotope internal standard [¹³C,¹⁵N₂]8-oxo-dGsn, and 1 to 2000 pg/μL isotope internal standard [¹⁵N₅]8-oxoGsn; preferably, the isotope internal standards have a concentration of 100 to 800 pg/μL.
 15. The kit according to claim 12[or 13], wherein the 10 mM ammonium acetate and methanol are 70% methanol+30% 10 mM ammonium acetate solution, and the solution after mixing has a pH of 3.5 to 4.0.
 16. The use according to claim 6, wherein the mammal is a human.
 17. The use according to claim 10, wherein the mammal is a human, and the product is a kit.
 18. The kit according to claim 13, wherein the kit comprises 8-oxoGsn standard, 10 mM ammonium acetate, methanol, 1 to 2000 pg/μL isotope internal standard [¹³C,¹⁵N₂]8-oxo-dGsn, and 1 to 2000 pg/μL isotope internal standard [¹⁵N₅]8-oxoGsn; preferably, the isotope internal standards have a concentration of 100 to 800 pg/μL.
 19. The kit according to claim 13, wherein the 10 mM ammonium acetate and methanol are 70% methanol+30% 10 mM ammonium acetate solution, and the solution after mixing has a pH of 3.5 to 4.0. 